Veränderungen des Plasmamembran-Proteoms in AP-2-depletierten HeLa-Zellen

Die Clathrin-vermittelte Endozytose (engl. clathrin-mediated endocytosis, CME) ermöglicht infolge einer Einstülpung der Plasmamembran und Ausbildung Protein-beschichteter Vesikel die zelluläre Aufnahme einer Vielzahl unterschiedlicher Membranproteine sowie ihrer Liganden. Zugleich nimmt die CME einen regulatorischen Einfluss auf die Zusammensetzung der Plasmamembran und wirkt sich in dieser Weise auf eine Vielzahl weiterer Zellprozesse aus. Insgesamt mehr als 50 ursprünglich aus dem Zytoplasma stammende Proteine sind im Zuge der CME an der Ausbildung der Vesikelhülle beteiligt, innerhalb derer der heterotetramere Adaptorkomplex AP-2 eine Schlüsselposition einnimmt. Im Rahmen der vorliegenden Arbeit wurde die CME unter Anwendung eines siRNA (engl. small interfering RNA, dt. kleine eingreifende RNS) -vermittelten Knockdowns (KD) der µ2-Untereinheit von AP-2 inhibiert und hieraus resultierende Veränderungen in Bezug auf die Endozytose unterschiedlicher Klassen von Membranproteinen untersucht. Hierbei stand die mittels quantitativer Massenspektrometrie erhobene Analyse des Plasmamembran-Proteoms µ2-depletierter HeLa-Zellen im Zentrum dieser Arbeit. Eine darüberhinausgehende Untersuchung einzelner Membranproteine erfolgte mittels Immunfluoreszenzmikroskopie und Durchflusszytometrie sowie unter Anwendung weiterer biochemischer und molekularbiologischer Methoden. Die gewonnenen Daten ergaben eine mindestens anderthalbfache Akkumulation mehr als der Hälfte aller identifizierten Proteine an der Plasmamembran µ2-depletierter HeLa-Zellen. Insbesondere zeigte sich eine starke Anreicherung endosomaler und lysosomaler Proteine sowie bestimmter Integrine. Funktionell äußerte sich Letzteres in einer deutlich beeinträchtigten Zellmigration. Darüber hinaus resultierte der im Rahmen dieser Arbeit angewandte µ2-KD in einer Plasmamembrananreicherung bekannter Tumormarker (CD73, CD164, CD302) sowie des mit soliden Tumoren assoziierten Metalloenzyms Carboanhydrase 9 (CA9), das einen möglichen Angriffspunkt für eine zielgerichtete antitumorale Therapie darstellt und die insgesamt stärkste Akkumulation aller Membranproteine erfuhr. Des Weiteren konnte das bislang nicht charakterisierte Membranprotein SMIM29 (engl. small integral membrane protein 29; Synonym: c6orf1) erstmals als ein Protein identifiziert werden, das mehrere funktionale Sortierungssignale aufweist und dessen Internalisierung mittels CME erfolgt. Ferner konnte neben der quantitativen Proteom-Analyse eine Hemmung der endozytotischen Gesamtleistung in AP-2-depletierten HeLa-Zellen nachgewiesen werden, während die Funktionsfähigkeit Clathrin-unabhängiger Endozytosewege (engl. clathrin-independent endocytosis, CIE) kaum beeinträchtigt war

Inhibition of clathrin-mediated endocytosis by knockdown of AP-2 leads to alterations in the plasma membrane proteome

In eukaryotic cells, clathrin-mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin-coated vesicles (CCVs) is AP-2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP-2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal-lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti-cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP-2 knockdown attenuated the global endocytic capacity, but clathrin-independent entry pathways were still operating, as indicated by persistent internalization of specific membrane-spanning and GPI-anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP-2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention.publishe

Inhibition of clathrin-mediated endocytosis by knockdown of AP-2 leads to alterations in the plasma membrane proteome

In eukaryotic cells, clathrin-mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin-coated vesicles (CCVs) is AP-2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP-2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal-lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti-cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP-2 knockdown attenuated the global endocytic capacity, but clathrin-independent entry pathways were still operating, as indicated by persistent internalization of specific membrane-spanning and GPI-anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP-2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention

Inhibition of clathrin‐mediated endocytosis by knockdown of AP

In eukaryotic cells, clathrin-mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin-coated vesicles (CCVs) is AP-2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP-2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal-lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti-cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP-2 knockdown attenuated the global endocytic capacity, but clathrin-independent entry pathways were still operating, as indicated by persistent internalization of specific membrane-spanning and GPI-anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP-2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention.publishe

Epidemiological trends and susceptibility patterns of bloodstream infections caused by Enterococcus spp. in six German university hospitals: a prospectively evaluated multicentre cohort study from 2016 to 2020 of the R-Net study group

Purpose: To analyse recent epidemiological trends of bloodstream infections (BSI) caused by Enterococcus spp. In adult patients admitted to tertiary care centres in Germany. Methods: Epidemiological data from the multicentre R-NET study was analysed. Patients presenting with E. faecium or E. faecalis in blood cultures in six German tertiary care university hospitals between October 2016 and June 2020 were prospectively evaluated. In vancomycin-resistant enterococci (VRE), the presence of vanA/vanB was confirmed via molecular methods. Results: In the 4-year study period, 3001 patients with BSI due to Enterococcus spp. were identified. E. faecium was detected in 1830 patients (61%) and E. faecalis in 1229 patients (41%). Most BSI occurred in (sub-) specialties of internal medicine. The pooled incidence density of enterococcal BSI increased significantly (4.0-4.5 cases per 10,000 patient days), which was primarily driven by VRE BSI (0.5 to 1.0 cases per 10,000 patient days). In 2020, the proportion of VRE BSI was > 12% in all study sites (range, 12.8-32.2%). Molecular detection of resistance in 363 VRE isolates showed a predominance of the vanB gene (77.1%). Conclusion: This large multicentre study highlights an increase of BSI due to E. faecium, which was primarily driven by VRE. The high rates of hospital- and ICU-acquired VRE BSI point towards an important role of prior antibiotic exposure and invasive procedures as risk factors. Due to limited treatment options and high mortality rates of VRE BSI, the increasing incidence of VRE BSI is of major concern

Increasing numbers and complexity of Staphylococcus aureus bloodstream infection - 14 years of prospective evaluation at a German tertiary care center with multi-center validation of findings

Objectives: Staphylococcus aureus bloodstream infection (SAB) is a common and severe infection. This study aims to describe temporal trends in numbers, epidemiological characteristics, clinical manifestations, and outcomes of SAB. Methods: We performed a post-hoc analysis of three prospective SAB cohorts at the University Medical Center Freiburg between 2006 and 2019. We validated our findings in a large German multi-center cohort of five tertiary care centers (R-Net consortium, 2017-2019). Time-dependent trends were estimated using Poisson or beta regression models. Results: We included 1,797 patients in the mono-centric and 2,336 patients in the multi-centric analysis. Overall, we observed an increasing number of SAB cases over 14 years (+6.4%/year and 1,000 patient days, 95%-CI: +5.1%-7.7%), paralleled by an increase in the proportion of community-acquired SAB (+4.9%/year (95%-CI: +2.1-7.8%), CA-SAB) and a decrease in the rate of methicillin-resistant-SAB (-8.5%/year (95%-CI: -11.2-(-5.6%)), MRSA-SAB). All of these findings were confirmed in the multi-center validation cohort (+6.2% cases per 1000 patient cases/year (95%-CI: -0.6-+12.6%), CA-SAB +8.7% (95%-CI: -1.2-+19.6%), MRSA-SAB -18.6% (95%-CI: -30.6-(-5.8%))). Moreover, we found an increasing proportion of patients with multiple risk factors for complicated/difficult-to-treat SAB (+8.5%/year, 95%-CI: +3.6-13.5%, p<0.001), alongside an overall higher level of comorbidities (Charlson comorbidity score +0.23 points/year, 95%-CI: +0.09-0.37, p=0.005). At the same time, the rate of deep-seated foci like osteomyelitis or deep-seated abscesses significantly increased (+6.7%, 95%-CI: +3.9-9.6%, p<0.001). A reduction of in-hospital mortality by 0.6% per year (95%-CI: 0.08-1%) was observed in the subgroup of patients with infectious diseases (ID) consultations. Conclusions: We found an increasing number of SAB combined with a significant increase in comorbidities and complicating factors in tertiary care centers. The resulting challenges in securing adequate SAB management in the face of high patient turnover will become an important task for physicians

Vancomycin-resistant Enterococcus faecium colonizing patients on hospital admission in Germany: prevalence and molecular epidemiology

Objectives: To analyse the rectal carriage rate and the molecular epidemiology of vancomycin-resistant Enterococcus faecium (VREfm) recovered from patients upon hospital admission. Methods: Adult patients were screened at six German university hospitals from five different federal states upon hospital admission for rectal colonization with VREfm between 2014 and 2018. Molecular characterization of VREfm was performed by WGS followed by MLST and core-genome MLST analysis. Results: Of 16350 patients recruited, 263 were colonized with VREfm, with increasing prevalence rates during the 5year study period (from 0.8% to 2.6%). In total, 78.5% of the VREfm were vanB positive and 20.2% vanA positive, while 1.2% harboured both vanA and vanB. The predominant ST was ST117 (56.7%) followed by ST80 (15%), ST203 (10.9%), ST78 (5.7%) and ST17 (3.2%). ST117/vanB VREfm isolates formed a large cluster of 96 closely related isolates extending across all six study centres and four smaller clusters comprising 13, 5, 4 and 3 isolates each. In contrast, among the other STs inter-regional clonal relatedness was rarely observed. Conclusions: To our knowledge, this is the largest admission prevalence and molecular epidemiology study of VREfm. These data provide insight into the epidemiology of VREfm at six German university hospitals and demonstrate the remarkable inter-regional clonal expansion of the ST117/vanB VREfm clone

Surveillance and Genomic Analysis of Third-Generation Cephalosporin-Resistant and Carbapenem-Resistant Klebsiella pneumoniae Complex in Germany

To analyse the epidemiology and population structure of third-generation cephalosporin-resistant (3GCR) and carbapenem-resistant (CR) Klebsiella pneumoniae complex isolates, patients were screened for rectal colonisation with 3GCR/CR K. pneumoniae complex on admission to six German university hospitals (2016-2019). Also collected were 3GCR/CR and susceptible K. pneumoniae isolates from patients with bloodstream infections (2016-2018). Whole-genome sequencing was performed followed by multilocus sequencing typing (MLST), core-genome MLST, and resistome and virulome analysis. The admission prevalence of 3GCR K. pneumoniae complex isolates during the 4-year study period was 0.8%, and 1.0 bloodstream infection per 1000 patient admissions was caused by K. pneumoniae complex (3GCR prevalence, 15.1%). A total of seven K. pneumoniae complex bloodstream isolates were CR (0.8%). The majority of colonising and bloodstream 3GCR isolates were identified as K. pneumoniae, 96.7% and 98.8%, respectively; the remainder were K. variicola and K. quasipneumoniae. cgMLST showed a polyclonal population of colonising and bloodstream isolates, which was also reflected by MLST and virulome analysis. CTX-M-15 was the most prevalent extended-spectrum beta-lactamase, and 29.7% of the colonising and 48.8% of the bloodstream isolates were high-risk clones. The present study provides an insight into the polyclonal 3GCR K. pneumoniae population in German hospitals